The origin and liver repopulating capacity of murine oval cells

The appearance of bipotential oval cells in chronic liver injury suggests the existence of hepatocyte progenitor/stem cells. To study the origin and properties of this cell population, oval cell proliferation was induced in adult mouse liver by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and a method for their isolation was developed. Transplantation into fumarylacetoacetate hydrolase (Fah) deficient mice was used to determine their capacity for liver repopulation. In competitive repopulation experiments, hepatic oval cells were at least as efficient as mature hepatocytes in repopulating the liver. In mice with chimeric livers, the oval cells were not derived from hepatocytes but from liver nonparenchymal cells. This finding supports a model in which intrahepatic progenitors differentiate into hepatocytes irreversibly. To determine whether oval cells originated from stem cells residing in the bone marrow, bone marrow transplanted wild-type mice were treated with DDC for 8 months and oval cells were then serially transferred into Fah mutants. The liver repopulating cells in these secondary transplant recipients lacked the genetic markers of the original bone marrow donor. We conclude that hepatic oval cells do not originate in bone marrow but in the liver itself, and that they have valuable properties for therapeutic liver repopulation.     

Comparison of hepatic properties and transplantation of Thy-1(+) and Thy-1(-) cells isolated from embryonic day 14 rat fetal liver

Thy-1, a marker of hematopoietic progenitor cells, is also expressed in activated oval cells of rat liver. Thy-1(+) cells are also in rat fetal liver and exhibit properties of bipotent hepatic epithelial progenitor cells in culture. However, no information is available concerning liver repopulation by Thy-1(+) fetal liver cells. Therefore, we isolated Thy-1(+) and Thy-1(-) cells from embryonic day (ED) 14 fetal liver and compared their gene expression characteristics in vitro and proliferative and differentiation potential after transplantation into adult rat liver. Fetal liver cells selected for Thy-1 expression using immunomagnetic microbeads were enriched from 5.2%-87.2% Thy-1(+). The vast majority of alpha fetoprotein(+), albumin(+), cytokine-19(+), and E-cadherin(+) cells were found in cultured Thy-1(-) cells, whereas nearly all CD45(+) cells were in the Thy-1(+) fraction. In normal rat liver, transplanted Thy-1(+) cells produced only rare, small DPPIV(+) cell clusters, very few of which exhibited a hepatocytic phenotype. In retrorsine-treated liver, transplanted Thy-1(+) fetal liver cells achieved a 4.6%-23.5% repopulation. In contrast, Thy-1(-) fetal liver cells substantially repopulated normal adult liver and totally repopulated retrorsine-treated liver. Regarding the stromal cell-derived factor (SDF)-1/chemokine (C-X-C motif) receptor 4 (CXCR4) axis for stem cell homing, Thy-1(+) and Thy-1(-) fetal hepatic epithelial cells equally expressed CXCR4. However, SDF-1alpha expression was augmented in bile ducts and oval cells in retrorsine/partial hepatectomy-treated liver, and this correlated with liver repopulation by Thy-1(+) cells. CONCLUSION: Highly enriched Thy-1(+) ED14 fetal liver cells proliferate and repopulate the liver only after extensive liver injury and represent a fetal hepatic progenitor cell population distinct from Thy-1(-) stem/progenitor cells, which repopulate the normal adult liver.     

A novel stem-cell population in adult liver with potent hematopoietic-reconstitution activity

A number of recent reports have documented that cells possessing hematopoietic-reconstitution ability can be identified and isolated from a variety of solid organs in the adult animal. In all studies to date, however, purified organ-derived stem cells demonstrate a diminished repopulating capacity relative to that of purified bone marrow-derived hematopoietic stem cells (BM HSCs). It has therefore been unclear whether organ-derived HSCs possess functional properties distinct from those of BM HSCs, or simply have not been purified to a comparable extent. Here we report the identification of a rare subset of cells in adult murine liver that possess potent blood-repopulating potential, approaching that of BM HSCs. The cells, isolated on the basis of dye-efflux activity and CD45 expression (termed CD45(+) liver side population [SP] tip cells), exhibit a surface phenotype similar to that of freshly isolated BM HSCs derived from normal adult animals, but are phenotypically distinct in that they do not express the stem-cell marker c-kit. Single-cell transplantation studies indicate that CD45(+) liver SP tip cells can be generated from BM HSCs, suggesting a relationship between stem-cell populations in the liver and bone marrow compartments. Overall, these studies have important implications for understanding extramedullary hematopoiesis, and may be relevant to current strategies aimed at inducing tolerance to transplanted organs.     

Fetal liver cell transplantation as a potential alternative to whole liver transplantation?

Because organ shortage is the fundamental limitation of whole liver transplantation, novel therapeutic options, especially the possibility of restoring liver function through cell transplantation, are urgently needed to treat end-stage liver diseases. Groundbreaking in vivo studies have shown that transplanted hepatocytes are capable of repopulating the rodent liver. The two best studied models are the urokinase plasminogen activator (uPA) transgenic mouse and the fumarylacetoacetate hydrolase (FAH)-deficient mouse, in which genetic modifications of the recipient liver provide a tissue environment in which there is extensive liver injury and selection pressure favoring the proliferation and survival of transplanted hepatocytes. Because transplanted hepatocytes do not significantly repopulate the (near-)normal liver, attention has been focused on finding alternative cell types, such as stem or progenitor cells, that have a higher proliferative potential than hepatocytes. Several sources of stem cells or stem-like cells have been identified and their potential to repopulate the recipient liver has been evaluated in certain liver injury models. However, rat fetal liver stem/progenitor cells (FLSPCs) are the only cells identified to date that can effectively repopulate the (near-)normal liver, are morphologically and functionally fully integrated into the recipient liver, and remain viable long-term. Even though primary human fetal liver cells are not likely to be routinely used for clinical liver cell repopulation in the future, using or engineering candidate cells exhibiting the characteristics of FLSPCs suggests a new direction in developing cell transplantation strategies for therapeutic liver replacement. This review will give a brief overview concerning the existing animal models and cell sources that have been used to restore normal liver structure and function, and will focus specifically on the potential of FLSPCs to repopulate the liver.     

Stem and progenitor cell systems in liver development and regeneration

The liver comprises two stem/progenitor cell systems: fetal and adult liver stem/progenitor cells. Fetal hepatic progenitor cells, derived from foregut endoderm, differentiate into mature hepatocytes and cholangiocytes during liver development. Adult hepatic progenitor cells contribute to regeneration after severe and chronic liver injuries. However, the characteristics of these somatic hepatic stem/progenitor cells remain unknown. Culture systems that can be used to analyze these cells were recently established and hepatic stem/progenitor cell‐specific surface markers including delta‐like 1 homolog (DLK), cluster of differentiation (CD) 13, CD133, and LIV2 were identified. Cells purified using antibodies against these markers proliferate for an extended period and differentiate into mature cells both in vitro and in vivo. Methods to force the differentiation of human embryonic stem and induced pluripotent stem (iPS) cells into hepatic progenitor cells have been recently established. We demonstrated that the CD13⁺CD133⁺ fraction of human iPS‐derived cells contained numerous hepatic progenitor‐like cells. These analyses of hepatic stem/progenitor cells derived from somatic tissues and pluripotent stem cells will contribute to the development of new therapies for severe liver diseases.     

Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150- fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.     

Hematopoietic progenitor/stem cells immortalized by Lhx2 generate functional hematopoietic cells in vivo

Hematopoietic stem cells (HSCs) are unique in their capacity to maintain blood formation following transplantation into immunocompromised hosts. Expansion of HSCs in vitro is therefore important for many clinical applications but has met with limited success because the mechanisms regulating the self-renewal process are poorly defined. We have previously shown that expression of the LIM-homeobox gene Lhx2 in hematopoietic progenitor cells derived from embryonic stem cells differentiated in vitro generates immortalized multipotent hematopoietic progenitor cell lines. However, HSCs of early embryonic origin, including those derived from differentiated embryonic stem cells, are inefficient in engrafting adult recipients upon transplantation. To address whether Lhx2 can immortalize hematopoietic progenitor/stem cells that can engraft adult recipients, we expressed Lhx2 in hematopoietic progenitor/stem cells derived from adult bone marrow. This approach allowed for the generation of immortalized growth factor-dependent hematopoietic progenitor/stem cell lines that can generate erythroid, myeloid, and lymphoid cells upon transplantation into lethally irradiated mice. When transplanted into stem cell-deficient mice, these cell lines can generate a significant proportion of circulating erythrocytes in primary, secondary, and tertiary recipients for at least 18 months. Thus, Lhx2 immortalizes multipotent hematopoietic progenitor/stem cells that can generate functional progeny following transplantation into lethally irradiated hosts and can long-term repopulate stem cell-deficient hosts.     

Stem cell-based regenerative opportunities for the liver: State of the art and beyond

The existing mismatch between the great demand for liver transplants and the number of available donor organs highlights the urgent need for alternative therapeutic strategies in patients with acute or chronic liver failure. The rapidly growing knowledge on stem cell biology and the intrinsic repair processes of the liver has opened new avenues for using stem cells as a cell therapy platform in regenerative medicine for hepatic diseases. An impressive number of cell types have been investigated as sources of liver regeneration: adult and fetal liver hepatocytes,intrahepatic stem cell populations,annex stem cells,adult bone marrow-derived hematopoietic stem cells,endothelial progenitor cells,mesenchymal stromal cells,embryonic stem cells,and induced pluripotent stem cells. All these highly different cell types,used either as cell suspensions or,in combination with biomaterials as implantable liver tissue constructs,have generated great promise for liver regeneration. However,fundamental questions still need to be addressed and critical hurdles to be overcome before liver cell therapy emerges. In this review,we summarize the state-of-the-art in the field of stem cell-based therapies for the liver along with existing challenges and future perspectives towards a successful liver cell therapy that will ultimately deliver its demanding goals.     

Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture

Although there have been numerous reports describing the isolation of liver progenitor cells from the adult liver, their exact origin has not been clearly defined; and the role played by mature hepatocytes as direct contributors to the hepatic progenitor cell pool has remained largely unknown. Here, we report strong evidence that mature hepatocytes in culture have the capacity to dedifferentiate into a population of adult liver progenitors without genetic or epigenetic manipulations. By using highly purified mature hepatocytes, which were obtained from untreated, healthy rat liver and labeled with fluorescent dye PKH2, we found that hepatocytes in culture gave rise to a population of PKH2-positive liver progenitor cells. These cells, liver-derived progenitor cells, which share phenotypic similarities with oval cells, were previously reported to be capable of forming mature hepatocytes, both in culture and in animals. Studies done at various time points during the course of dedifferentiation cultures revealed that hepatocytes rapidly transformed into liver progenitors within 1 week through a transient oval cell-like stage. This finding was supported by lineage-tracing studies involving double-transgenic AlbuminCreXRosa26 mice expressing β-galactosidase exclusively in hepatocytes. Cultures set up with hepatocytes obtained from these mice resulted in the generation of β-galactosidase-positive liver progenitor cells, demonstrating that they were a direct dedifferentiation product of mature hepatocytes. Additionally, these progenitors differentiated into hepatocytes in vivo when transplanted into rats that had undergone retrorsine pretreatment and partial hepatectomy. CONCLUSION: Our studies provide strong evidence for the unexpected plasticity of mature hepatocytes to dedifferentiate into progenitor cells in culture, and this may potentially have a significant effect on the treatment of liver diseases requiring liver or hepatocyte transplantation.     

细胞移植疗法在肝病中的研究与应用

人们对使用细胞移植疗法恢复肝脏功能的前景寄予厚望,期待其最终能够代替肝脏移植手术.众多学者对成人成熟肝细胞,不同来源干细胞的再生能力方面进行了大量研究,特别是间充质干细胞来源的肝细胞移植疗法在恢复肝脏再生方面的效果在动物模型实验中取得令人鼓舞的结果.本文将对近年来细胞移植治疗各种肝病的机制研究、动物实验以及临床试验等方...     

New cell surface markers for murine fetal hepatic stem cells identified through high density complementary DNA microarrays

Isolation of hepatic stem cells from the adult liver (AL) has not yet been achieved due to the lack of specific cell surface markers. To identify new surface markers for hepatic stem cells, we analyzed differences in the gene expression profile of embryonic day (ED) 13.5 fetal liver stem/progenitor cells (FLSPC) versus AL by complementary DNA (cDNA) microarray technology. Using FLSPC purified to >90% by immunomagnetic selection for E-cadherin and high density (27k) mouse cDNA microarrays, we identified 474 genes that are more strongly expressed in FLSPC (FLSPC-up genes) and 818 genes that are more strongly expressed in AL (AL-up genes). The most highly overrepresented gene ontology (GO) categories for FLSPC-up genes are nucleus, cellular proliferation, and cell cycle control. AL-up genes are overrepresented for genes in metabolic pathways for specific hepatic functions. We identified 24 FLSPC-up gene surface markers and 69 AL-up gene surface markers. Western blot studies confirmed the expression of the FLSPC-up gene neighbor of Punc E11 (Nope) in fetal liver, but expression was not detectable in AL. Immunohistochemistry, confocal microscopy, and fluorescence-activated cell sorting (FACS) analysis of fetal liver demonstrated that Nope is specifically expressed on the surface of FLSPC within the fetal liver. CONCLUSION: This is the first microarray study to analyze the specific gene expression profile of purified murine FLSPC. Our analysis identified 24 new/potential cell surface markers for murine fetal hepatic stem cells, of which Nope may be particularly useful in future studies to identify, characterize and isolate hepatic stem cells from the AL.     

Long-term extensive expansion of mouse hepatic stem/progenitor cells in a novel serum-free culture system

BACKGROUND & AIMS: The liver has high regenerative potential. We attempted to establish a novel culture system for extensive expansion of fetal mouse hepatic stem/progenitor cells and to characterize cultured cells. METHODS: Hepatic spheroids collected from 6-day floating cultures were cultured on collagen-coated dishes in serum-free conditions in medium containing growth factors. Cultured cells were mainly characterized by immunocytochemistry and flow cytometry or transplanted into adult mice. RESULTS: Approximately 400 expanding hepatic spheroids were generated from every 1 x 10(6) fetal liver cells. Subsequently, highly replicative colonies were subcultured with maintaining colony formation on collagen-coated dishes. These colonies consisted of small immature alpha-fetoprotein-positive cells and hepatocytic and cholangiocytic lineage-committed cells. The immature alpha-fetoprotein-positive cells could be expanded in a reproducible manner at least 5 x 10(5)-fold (which involved at least 30 passages over >6 months) without losing differentiation potential. Flow cytometric analysis showed that all cultured cells expressed CD49f, but not CD34, Thy-1, c-kit, or CD45. Nearly 15% of the cells expressed Sca-1, and approximately 5%-20% of the cells were side population cells. Both sorted side population cells and Sca-1-positive cells (especially side population cells) produced a large number of alpha-fetoprotein-positive cells and lineage-committed cells. Expanded cells had bidirectional differentiation potential and improved serum albumin levels in mice with severe liver damage. CONCLUSIONS: Long-term extensive expansion of transplantable hepatic stem/progenitor cells was reproducibly achieved in a novel serum-free culture system. Moreover, this culture system yielded side population and Sca-1-positive cell populations that included hepatic stem/progenitor cells with differentiation and proliferation properties.     

The diversity and plasticity of adult hepatic progenitor cells and their niche

The liver is a unique organ for homoeostasis with regenerative capacities. Hepatocytes possess a remarkable capacity to proliferate upon injury; however, in more severe scenarios liver regeneration is believed to arise from at least one, if not several facultative hepatic progenitor cell compartments. Newly identified pericentral stem/progenitor cells residing around the central vein is responsible for maintaining hepatocyte homoeostasis in the uninjured liver. In addition, hepatic progenitor cells have been reported to contribute to liver fibrosis and cancers. What drives liver homoeostasis, regeneration and diseases is determined by the physiological and pathological conditions, and especially the hepatic progenitor cell niches which influence the fate of hepatic progenitor cells. The hepatic progenitor cell niches are special microenvironments consisting of different cell types, releasing growth factors and cytokines and receiving signals, as well as the extracellular matrix (ECM) scaffold. The hepatic progenitor cell niches maintain and regulate stem cells to ensure organ homoeostasis and regeneration. In recent studies, more evidence has been shown that hepatic cells such as hepatocytes, cholangiocytes or myofibroblasts can be induced to be oval cell-like state through transitions under some circumstance, those transitional cell types as potential liver-resident progenitor cells play important roles in liver pathophysiology. In this review, we describe and update recent advances in the diversity and plasticity of hepatic progenitor cell and their niches and discuss evidence supporting their roles in liver homoeostasis, regeneration, fibrosis and cancers.     

Expansion of hematopoietic stem cells in the developing liver of a mouse embryo

The activity of hematopoietic stem cells in the developing liver of a C57BL/6 mouse embryo was quantified by a competitive repopulation assay. Different doses of fetal liver cells at days 11 to 18 of gestation were transplanted into irradiated mice together with 2 x 10(5) adult bone marrow cells. A long-term repopulation in myeloid-, B-cell, and T-cell lineage by fetal liver cells was evaluated at 20 weeks after transplantation. At day 12 of gestation multilineage repopulating activity was first detected in the liver as 50 repopulating units (RU) per liver. The number of RU per liver increased 10-fold and 33-fold by day 14 and day 16 of gestation, and decreased thereafter, suggesting a single wave of stem cell development in the fetal liver. A limiting dilution analysis revealed that the frequency of competitive repopulating units (CRU) in fetal liver cells at day 12 of gestation was similar to that at day 16 of gestation. Because of an increase of total fetal liver cell number, the absolute number of CRU per liver from days 12 to 16 of gestation increased 38-fold. Hence, the mean activity of stem cells (MAS) that is given by RU per CRU remained constant from days 12 to 16 of gestation. From these data we conclude that hematopoietic stem cells expand in the fetal liver maintaining their level of repopulating potential.     

Primary endothelial cells isolated from the yolk sac and para-aortic splanchnopleura support the expansion of adult marrow stem cells in vitro

The embryonic origin and development of hematopoietic and endothelial cells is highly interdependent. We hypothesized that primary endothelial cells from murine yolk sac and para-aortic splanchnopleura (P-Sp) may possess the capacity to expand hematopoietic stem cells (HSCs) and progenitor cells ex vivo. Using Tie2-GFP transgenic mice in combination with fluorochrome-conjugated monoclonal antibodies to vascular endothelial growth factor receptor-2 (Flk1) and CD41, we have successfully isolated pure populations of primary endothelial cells from 9.5-days after coitus (dpc) yolk sac and P-Sp. Adult murine bone marrow Sca-1+c-Kit+lin- cells were cocultured with yolk sac or P-Sp Tie2-GFP+Flk-1+CD41- endothelial cell monolayers for 7 days and the total number of nonadherent cells increased 47- and 295-fold, respectively, and hematopoietic progenitor counts increased 9.4- and 11.4-fold, respectively. Both the yolk sac and P-Sp endothelial cell cocultures facilitated long-term (> 6 months) HSC competitive repopulating ability (2.8- to 9.8-fold increases, respectively). These data suggest that 9.5-dpc yolk sac- and P-Sp-derived primary Tie2-GFP+Flk-1+CD41- endothelial cells possess the capacity to expand adult bone marrow hematopoietic progenitor cell and HSC repopulating ability ex vivo.     

Identifying and tracking neural stem cells

Hematopoietic stem cells, unlike neural stem cells, can be readily identified and isolated from developing and adult cell populations using positive and negative selection criteria. Isolating stem cells and progenitor cells from neural tissue has been more difficult because of difficulties in separating cells in solid tissue, the limited numbers of stem cells that persist in the adult, and the paucity of rigorously characterized markers. Nevertheless, strategies that have worked successfully in hematopoietic stem cell isolation can be adapted to isolate multiple classes of stem and progenitor cells from neural tissue. Neural stem cells also share cellular and molecular properties with other stem cell populations that may serve as surrogate identifiers of multipotentiality. Such potential markers are described. Unlike hematopoietic stem cells, tracking neural cells after transplantation is both necessary and more difficult. It will therefore be necessary to develop invasive and non-invasive strategies to follow transplanted cells and develop useful quantifiable readouts. Some potential strategies are described and current results are discussed.     

Hepatocyte transplantation: Studies in preclinical models

Summary Transplantation of allogeneic or genetically modified autologous hepatocytes may be an alternative to whole-liver transplantation for the treatment of hereditary metabolic liver diseases. Human hepatocytes have already been transplanted in patients, demonstrating the safety and feasibility of both approaches. Although a few cases of allogeneic transplantation have resulted in long-term engraftment and function, only a partial and transient correction of the disease was achieved. This may partly result from a lack of proliferation of transplanted cells. In rodents, transplanted hepatocytes do not proliferate in adult quiescent livers and repopulate recipient livers only when they display a proliferative advantage over resident hepatocytes. Most of these models are not transposable to humans, however. Our aim is to develop preclinical approaches to hepatocyte transplantation in nonhuman primates. We have defined a strategy that increases the engraftment efficiency of transplanted hepatocytes by inducing their proliferation together with that of resident hepatocytes. We have also immortalized simian fetal hepatic progenitor cells and shown that these cells do not proliferate in situ after transplantation into the livers of immunodeficient mice. By contrast early human hepatoblasts repopulate mouse livers more efficiently. However, if we consider the number of cells to be transplanted (one to several billion), the means of expanding and differentiating stem or progenitor cells other than hepatocytes will have to be determined prior to envisaging treating patients. Presented at the 42nd Annual Meeting of the SSIEM, Paris, 6–9 September, 2005. Competing interests: None declared     

Repopulation of rat liver by fetal hepatoblasts and adult hepatocytes transduced ex vivo with lentiviral vectors

Recent studies have shown that nondividing primary cells, such as hepatocytes, can be efficiently transduced in vitro by human immunodeficiency virus-based lentivirus vectors. Other studies have reported that, under certain conditions, the liver can be repopulated with transplanted hepatocytes. In the present study, we combined these procedures to develop a model system for ex vivo gene therapy by repopulating rat livers with hepatocytes and hepatoblasts transduced with a lentivirus vector expressing a reporter gene, green fluorescent protein (GFP). Long-term GFP expression in vivo (up to 4 months) was achieved when the transgene was driven by the liver-specific albumin enhancer/promoter but was silenced when the cytomegalovirus (CMV) enhancer/promoter was used. Transplanted cells were massively amplified ( approximately 10 cell doublings) under the influence of retrorsine/partial hepatectomy, and both repopulation and continued transgene expression in individual cells were documented by dual expression of a cell transplantation marker, dipeptidyl peptidase IV (DPPIV), and GFP. In this system, maintenance or expansion of the transplanted cells did not depend on expression of the transgene, establishing that positive selection is not required to maintain transgene expression following multiple divisions of transplanted, lentivirus-transduced hepatic cells. In conclusion, fetal hepatoblasts (liver stem/progenitor cells) can serve as efficient vehicles for ex vivo gene therapy and suggest that liver-based genetic disorders that do not shorten hepatocyte longevity or cause liver damage, such as phenylketonuria, hyperbilirubinemias, familial hypercholesterolemia, primary oxalosis, and factor IX deficiency, among others, might be amenable to treatment by this approach.     

Hematopoietic capability of CD34+ cord blood cells: a comparison with CD34+ adult bone marrow cells

The characteristics of hematopoietic progenitor and stem cell (HPC/HSC) populations in mammals vary according to their ontogenic stage. In humans, HPC/HSCs from umbilical cord blood (CB) are increasingly used as an alternative to HPC/HSCs from adult bone marrow (BM) for the treatment of various hematologic disorders. How the hematopoietic activity of progenitor and stem cells in CB differs from that in adult BM remains unclear, however. We compared CD34+ cells, a hematopoietic cell population, in CB with those in adult BM using phenotypic subpopulations analyzed by flow cytometry, the colony-forming activity in methylcellulose clonal cultures, and the repopulating ability of these cells in NOD/Shi-scid (NOD/SCID) mice. Although the proportion of CD34+ cells was higher in adult BM than in CB mononuclear cells, the more immature subpopulations, CD34+ CD33- and CD34+ CD38- cells, were present in higher proportions in CD34+ CB cells. Clonal culture assay showed that more multipotential progenitors were present in CD34+ CB cells. When transplanted into NOD/SCID mice. CD34+ adult BM cells could not reconstitute human hematopoiesis in recipient BM, but CD34+ CB cells achieved a high level of engraftment, indicating that CD34+ CB cells possess a greater repopulating ability. These results demonstrated that human hematopoiesis changes with development from fetus to adult. Furthermore, CD34+ CB cells contained a greater number of primitive hematopoietic cells, including HSCs, than did adult BM, suggesting the usefulness of CD34+ CB cells not only as a graft for therapeutic HSC transplantation but also as a target cell population for ex vivo expansion of transplantable HSCs and for gene transfer in gene therapy.